Abstract

This study reveals the essence of ligand recognition mechanisms by which calmodulin (CaM) controls a variety of Ca2+ signaling processes. We study eight forms of calcium-loaded CaM each with distinct conformational states. Reducing the structure to two degrees of freedom conveniently describes main features of the conformational changes of CaM via simultaneous twist-bend motions of the two lobes. We utilize perturbation-response scanning (PRS) technique, coupled with molecular dynamics simulations. PRS is based on linear response theory, comprising sequential application of directed forces on selected residues followed by recording the resulting protein coordinates. We analyze directional preferences of the perturbations and resulting conformational changes. Manipulation of a single residue reproduces the structural change more effectively than that of single/pairs/triplets of collective modes of motion. Our findings also give information on how the flexible linker acts as a transducer of binding information to distant parts of the protein. Furthermore, by perturbing residue E31 located in one of the EF hand motifs in a specific direction, it is possible to induce conformational change relevant to five target structures. Independently, using four different pKa calculation strategies, we find this particular residue to be the charged residue (out of a total of 52), whose ionization state is most sensitive to subtle pH variations in the physiological range. It is plausible that at relatively low pH, CaM structure is less flexible. By gaining charged states at specific sites at a pH value around 7, such as E31 found in the present study, local conformational changes in the protein will lead to shifts in the energy landscape, paving the way to other conformational states. These findings are in accordance with Fluorescence Resonance Energy Transfer (FRET) measured shifts in conformational distributions towards more compact forms with decreased pH. They also corroborate mutational studies and proteolysis results which point to the significant role of E31 in CaM dynamics.

Received 15 July 2011Accepted 26 September 2011Published online 19 October 2011

Acknowledgments:

This work was supported by the Scientific and Technological Research Council of Turkey Project (Grant 110T624). A.O.A. acknowledges Youssef Jameel Scholarship for her Ph.D. studies.

Article outline:I. INTRODUCTIONII. MATERIALS AND METHODSA. ProteinsB. Perturbation response scanningC. Molecular dynamics simulationsD. Modal analysisE. Overlap of target structuresF. pKa calculationsIII. RESULTSA. A survey of the protein structuresB. Directionality matters for conformational change preferences of CaMC. Can conformational changes of CaM be described by slow modes of motion?D. A twist and a bend overcome a local free energy barrierE. E31 is a signaling residue for global communication in CaM through the linkerF. Degree of ionization calculations identify E31 as a proton uptake/release site at physiological pH rangeIV. DISCUSSION